Morphological and molecular data on acuariid nematodes in European great cormorants (Phalacrocorax carbo sinensis) and pygmy cormorants (Microcarbo pygmaeus)

The family Acuariidae is a speciose group of parasitic nematodes, infecting mostly birds as definitive hosts. This study focused on the characterization of two species of acuariids, collected in two different species of piscivorous birds, the European great cormorant Phalacrocorax carbo sinensis from Italy, and the pygmy cormorant Microcarbo pygmaeus from Israel. Parasites were analyzed using light and scanning electron microscopy and by amplification and sequencing of the 28S rDNA. The results of morphological and molecular analyses showed that Ph. carbo sinensis was infected by the acuariid Syncuaria squamata (12 females) and Cosmocephalus obvelatus (1 female), whereas M. pygmaeus was infected by C. obvelatus (2 males, 12 females). The present results provide new data on the distribution of acuariid parasites of piscivorous birds, the first report of Acuariidae in Israel, and the first molecular data on S. squamata and C. obvelatus, which will be useful in future epidemiological and phylogenetic studies of these widely distributed, but less molecularly studied parasites.


Materials and methods
Nematodes were extracted from the intestine of 1 Ph.carbo sinensis found dead (Emilia Romagna, Italy in 2023), and from the gastric mucosa of 3 Microcarbo pygmaeus (2 from Mevo Hama and 1 from Kfar Ruppin, Israel in 2021) shot and processed fresh.For the birds from Israel all experimental protocols were approved under permit 2020/42686 from the Israel Nature and Parks Authority; all methods were carried out in accordance with relevant guidelines and regulations; all methods are reported in accordance with ARRIVE guidelines (https:// arriv eguid elines.org).In both cases the worms were carefully washed in saline and cleaned with a small brush, then preserved in 70% ethanol for downstream analyses.Few specimens from each locality were also preserved in 10% neutral buffered formalin for SEM studies.
The nematodes were observed under a dissection microscope to first evaluate gross morphology and measure the total length (TL), then under a light microscope (Leica Microsystems, Wetzlar, Germany) with the aid of a digital Nikon DS-Fi1 camera and image-acquisition software (Nikon Nis-Elements D3.0).A section of each worm was removed for DNA extraction (central 5 mm, devoided of taxonomically informative features).Anterior and posterior portions were then clarified in Amman's lactophenol to measure internal structures by light microscope.Species identification was carried out following 1, 13,14 .
For SEM, anterior and posterior portions of the worms were dehydrated through a graded ethanol series, subjected to critical point drying, sputter-coated with gold palladium, and observed using a Phenom XL G2 Desktop SEM (Thermo Fisher Scientific, Eindhoven, The Netherlands) operating at 5 kV.
For the molecular analysis, the genomic DNA from 20 specimens (13 from Italy and 7 from Israel) was extracted using PureLink® Genomic DNA Kit (Life Technologies, Carlsbad, California) following the manufacturer's instructions.The amplification of the D1-D3 variable region of the 28S rDNA was performed with primers U178_f (5′-GCA CCC GCT GAA YTT AAG -3′) and L1642_r (5′-CCA GCG CCA TCC ATT TTC A-3′) 15 .The thermal cycler program (Tpersonal, Biometra) was 40 cycles of 30 s at 94 °C, 30 s at 52 °C and 2 min at 72 °C, preceded by a denaturation step at 94 °C for 2 min and followed by an extended elongation step at 72 °C for 10 min.The PCR products were electrophoresed on 1% agarose gel stained with SYBR® Safe DNA Gel Stain (Thermo Fisher Scientific, Carlsbad, California) in 0.5X TBE.By NucleoSpin Gel and PCR Cleanup (Mackerey-Nagel, Düren, Germany) the amplicons were purified and sequenced with an ABI 3730 DNA analyzer (StarSEQ Mainz, Germany).The DNA trace files were assembled with ContigExpress (VectorNTI Advance 11 software, Invitrogen, Carlsbad, California) and the consensus sequences were compared with previously published data by BLAST tools (https:// blast.ncbi.nlm.nih.gov/ Blast.cgi).Multiple sequences alignments were constructed using BioEdit 7.2.5 together with some of the sequences reported by Mutafchiev et al. 3 .Pairwise distance, and maximum likelihood (ML) tree (GTR + G, bootstrap of 1,000 replicates) were obtained by MEGA 7.

Results
A total of 13 acuariid nematodes (all females) were recovered from Ph. carbo sinensis and morphologically identified as Syncuaria squamata (Linstow, 1883) except one identified as Cosmocephalus obvelatus (Creplin, 1825) Seurat, 1919, while 14 acuariids (2 males, 12 females) were found in M. pygmaeus (intensity ranging from 1 to 10) and morphologically identified as C. obvelatus.Morphometric data of the specimens analyzed are reported in Tables 1 and 2 and are expressed in micrometres (µm), unless otherwise stated.
Among the 7 specimens collected in Israel, only one gave a readable pherograms, probably due to preservation issues; the similarity was 99.4% with C. jaenschi (OP455796 16 ).The p-distance analysis revealed that all the Syncuaria sequences obtained in this study were identical to each other and showed a 0.8% distance from S. sagittata.This confirms that they do not belong to the latter species, as observed through morphological identification, but rather to S. squamata, for which no sequences are available in GenBank.In fact, in the Maximum Likelihood (ML) tree (Fig. 3), our sequences form a well-supported cluster, distinctly separated from the cluster (98% bootstrap) formed by S. sagittata and D. decorata.
Concerning the Cosmocephalus sp., the two specimens (one from Italy and one from Israel) were identical to each other with a p-distance of 1.6% with C. jaenschi (OP455795 16 ) but 0% with OP455796 (C.jaenschi 16 ).These results coupled with the morphological observation allowed to state that our species was not C. jaenschi but C. obvelatus (no sequences available in GB) and move the specimen from sprat of Bennett et al. 16 in the latter species; the ML tree confirmed this observation (100% bootstrap) (Fig. 3).

Discussion
The genus Syncuaria Gilbert, 1927 comprises eight species parasitizing members of the Ciconiiformes and one species specific of grebes 17 .The genera Skrjabinocara Kurashvili, 1940 and Decorataria Sobolev, 1949 have been considered as synonyms of Syncuaria 13,18,19 , although other authors have retained the validity of the three genera 2,3,20 .
The species S. squamata has been reported from the proventriculus and gizzard of different cormorant species (Ph.Carbo (Linnaeus), Ph. auritus (Lesson, 1831), Ph. brasilianus (Gmelin, 1789) and M. pygmaeus) and from Accipitriformes 1,21 .It was originally described as Filaria squamata by Linstow 22 , then redescribed as Skrjabinocara squamata by Kurochkin 10 , and finally assigned to the genus Syncuaria by Wong et al. 13 .This species is widely distributed and reported from different areas of Europe, Asia, and America 1, 23 .
In the definitive host, prevalence and intensity values can be highly variable: Kanarek and Rolbiecki 24 found that S. squamata infection was heavier in the immature birds (84.6%, 40.9 worms, and 1-120 worms) than in the adults (3.7%, 1 worm).These differences may suggest either a possible acquired resistance or an ontogenetic dietary shift of the cormorants.Moravec and Scholz 23 found prevalence values of 15% and 20% and intensity values of 1-12 (mean = 4) and 1-4 (mean = 2) in Ph. carbo sinensis from South Bohemia and South Moravia, respectively.In Italy, S. squamata has already been reported from great cormorants with a prevalence around 20% and intensity values ranging from 1 to 10 25 .In Ph. carbo sinensis examined during the present investigation, intensity of S. squamata was 14.This overall low intensity may be related to possible mechanisms of competitive interactions with other species of parasitic helminths, as suggested by Dezfuli et al. 25 ; in fact, in the same Ph.carbo sinensis analyzed in the present work, high intensity values of the anisakid nematodes Contracaecum rudolphii A and Contracaecum rudolphii B were also observed 26 ; the same M. pygmaeus analyzed in the present work, were infected also by Amirthalingamia macracantha (Joyeux and Baer 1935) (Cestoda) 27 .
Interestingly, in our study only female specimens were recovered.This finding is in accordance with the observation of Monteiro et al. 21, who found only one male out of 9 S. squamata recovered from Ph. brasilianus.Morphological features, as observed in light and scanning electron microscopy, and morphometry (Table 1) are in accordance with previous studies 1, 13,21,23,24,28 , although variability in the shape of deirids (both bifid and trifid) has been reported by some of these authors 21,23 .The only Syncuaria available in GenBank is the species S. sagittata, which in the ML tree is clearly separated from S. squamata described in the present study; the two species also show different morphological features.Particularly, S. sagittata was previously known as Desportesius sagittatus (Rudolphi, 1809) 13,29 and was subsequently assigned to the genus Syncuaria based on phylogenetic analysis 3  (Mutafchiev et al. 2020); this species is found in the gizzard of different birds (Ardea purpurea Linnaeus, 1766, Ciconia nigra Linnaeus, 1758, Nycticorax nycticorax Linnaeus, 1758) 1 and is clearly distinguishable from S. squamata by its overall smaller size and by the morphology of anterior end, characterized by recurrent cordons reaching approximately half of the muscular oesophagus 30 .Therefore, our molecular data further support the separation between these two congeneric species.
The genus Cosmocephalus includes seven species 14 , all parasitic in aquatic birds.Particularly, the species C. obvelatus shows a cosmopolitan distribution and low specificity, being reported from a wide range of bird species 1,7 , including several species of cormorants (Phalacrocoracidae).In other groups of bird parasites (e.g. in Anisakidae of the genus Contracaecum Railliet & Henry, 1912), the use of molecular data has helped to reveal hidden genetic diversity 26 ; however, as mentioned earlier, few sequence data of acuariids are available in public databases: for the genus Cosmocephalus, such data are available only for the species C. jaenschi.Nevertheless, the wide distribution of this species has also been associated to its ecology and to the wide-ranging ability of its hosts 7,31 .
Cosmocephalus obvelatus was previously reported in Ph. carbo sinensis from the European continent 32,33 and in M. pygmaeus 1 ; our study reports for the first time the occurrence of this nematode species in birds from Italy and Israel.Unidentified Cosmocephalus sp. was also reported in M. pygmaeus from Indonesia 34 however morphological details provided are insufficient to confirm its specific identity.Another congeneric species reported from M. pygmaeus is C. jaenschi, which differed from C. obvelatus mainly for having tricuspid deirids (but bicuspid deirids have also been occasionally described, see Presswell and Bennett 35 and different spicule morphometry.In particular, the size range of spicules from our specimens overlaps with that reported in recent detailed redescription of C. obvelatus 14,31 and in older descriptions from Europe 1,36 , while it differs from the size range reported for C. jaenschi 14,35 .Both species are described as having an appendage on the distal end of the left spicule; however, in C. jaenschi this appendage appears more prominent than in C. obvelatus 14 .Furthermore, in our male specimens, the proximal pair of precloacal papillae lies slightly external to the distribution of the other precloacal papillae, a feature already reported for C. obvelatus 31 . Interestingly, C. jaenschi has been reported mostly, although not exclusively, from the southern hemisphere 16,35,[37][38][39] .Indeed, in the current study, the phylogenetic analysis revealed that the larval specimen identified as C. jaenschi (OP455796) by Bennet et al. 16 in Sprattus antipodum (Hector, 1872) from New Zealand actually belongs to the species C. obvelatus.This conclusion is supported by a p-distance of 0% and a bootstrap value of 100%.
At present, morphological information on larval stages of most acuariid species is insufficient to allow their identification to the species level, further supporting the possibility of a misidentification.
The morphology of male and female C. obvelatus from our study are in accordance with other light and scanning electron microscopy studies of specimens from different hosts 7,14,31,40-42 .

Figure 3 .
Figure 3. Maximum Likelihood (GTR model) tree based on 28S rDNA sequences newly generated together with other acuariids retrieved from GenBank.The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.The analysis involved 25 nucleotide sequences.

Table 1 .
Morphometric data from adult male and female Syncuaria squamata examined in this study, along with a comparison to previously documented descriptions.na = not available.

Table 2 .
Morphometric data from adult male and female Cosmocephalus obvelatus examined in this study, along with a comparison to previously documented descriptions.na = not available.